foods Article Use of Invasive Green Crab Carcinus maenas for Production of a Fermented Condiment Delaney M. Greiner, Denise I. Skonberg, Lewis B. Perkins and Jennifer J. Perry * School of Food and Agriculture, The University of Maine, Orono, ME 04469, USA; [email protected] (D.M.G.); [email protected] (D.I.S.); [email protected] (L.B.P.) * Correspondence: [email protected]; Tel.: +1-207-581-2940 Abstract: To control the population of an invasive species of green crab, we investigated the feasibility of producing a fermented crab condiment. Commercial fermented fish condiments were tested to assess variability in the marketplace and to identify targets for lab-fermented sauces. Finely chopped crab was combined with 100 mg g−1, 200 mg g−1, or 300 mg g−1 NaCl, and spontaneously fermented for up to 120 days. Chromatographic analysis revealed that histamine content was not a safety concern as all treatments were below the current U.S. legal threshold (50 mg 100 mL−1). The majority of microbial and physicochemical properties measured within salt level (proteolytic bacterial population, total volatile basic nitrogen (TVBN), amine nitrogen, water activity, moisture, and biogenic amines) were statistically unchanged between days 60 and 120 of fermentation, suggesting that most of the biochemical changes happened early in the fermentation. While the production of a fermented condiment was successful and could represent an opportunity for the valorization of this invasive species, additional work is needed to accelerate the process and further understand the dynamics of the early fermentation stages. Citation: Greiner, D.M.; Skonberg, D.I.; Perkins, L.B.; Perry, J.J. Use of Keywords: Carcinus maenas; histamine; fermentation; fish sauce; green crab Invasive Green Crab Carcinus maenas for Production of a Fermented Condiment. Foods 2021, 10, 659. https://doi.org/10.3390/ 1. Introduction foods10040659 The European green crab (Carcinus maenas) is an extremely aggressive invasive species established on North America’s east and west coasts [1,2]. The green crab preys on Academic Editors: Marios Mataragas commercially relevant clam and mussel species, which has adversely affected coastal and Loulouda Bosnea regions ecologically and economically. This predation has decreased soft-shelled clam populations on the east coast of the United States by 40%, representing a loss of $22.6 million Received: 5 February 2021 per year in sales [2]. Adverse effects of this invasive species are not limited to predation. Accepted: 16 March 2021 Published: 24 March 2021 Mature and juvenile green crabs damage important ecosystems by eating eelgrass and digging through the sediment surrounding eelgrass [3]. These eelgrass beds are a critical Publisher’s Note: MDPI stays neutral habitat, serving as nurseries for juvenile fish, providing protection from native predators, with regard to jurisdictional claims in and acting as an essential food resource for diverse species including waterfowl [4]. The published maps and institutional affil- presence of green crabs also poses a threat to the American lobster population through iations. competition over prey [5]. Unfortunately, hard shelled green crabs are difficult to use in the culinary industry because of difficulties removing meat from this diminutive species. One opportunity to control this species that has been investigated is the establishment of a soft-shell fishery. This fishery could help exert local control over the population of green crab and is con- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. sidered low barrier to entry due to the use of existing fishing materials and the lack of This article is an open access article trapping limits on this species [1]. In Italy, there are mature soft-shell green crabs selling −1 −1 distributed under the terms and for as much as €51.14 kg , or about $27 USD lb [1]. Other current uses for the crabs conditions of the Creative Commons include composting and bait [6], but the establishment of a soft-shelled industry represents Attribution (CC BY) license (https:// the highest value opportunity for fishers. creativecommons.org/licenses/by/ Pre-molt green crabs are easily identified and can be stored live until suitable for sale 4.0/). in a soft-shell market. However, a significant portion of the trapped crabs are not suitable Foods 2021, 10, 659. https://doi.org/10.3390/foods10040659 https://www.mdpi.com/journal/foods Foods 2021, 10, 659 2 of 11 for live storage. Although this excess biomass could be sold as bait or composted, the establishment of an edible use for the excess material would command a higher price for fishers. This additional valorization of excess biomass further incentivizes fishers and makes the system more sustainable [7]. Fermented fish sauce is a clear liquid, brown in color, with a distinct fish flavor [8], which, in its simplest form, consists of the liquid resulting from the spontaneous fermenta- tion of salted whole anchovies and other fish species [9]. The fermentation of a fish sauce typically ranges between 6 and 18 months [8,10], though duration depends on ambient temperature and local preferences. Fermented seafood sauce is historically a widely used condiment in a large number of cultures, particularly those with Asian influence, and is becoming more common in American kitchens [11]. Anchovies are commonly used in fish sauce manufacture because of their low value and abundance of protein, with the Peruvian anchovy having up to 20% protein [12]. Al- though anchovies are the most commonly used substrate, diverse species including tilapia, sardines, silver barb fish, and freshwater crab have been used in fermented condiment production [13–17]. Carcinus maenas has a protein content of about 17% [18], which could be fermented as a way to utilize this valuable protein in the culinary industry without the need to separate the shell and meat. Additionally, this process is relatively low input with little capital investment needed, making this product accessible for small-scale processing operations or cottage producers. The purpose of this work was to assess the physicochemi- cal properties of existing commercial fish sauce products to establish baseline values for a novel, fermented seafood condiment, and to assess the feasibility of using whole green crab as the substrate for such a product. 2. Materials and Methods 2.1. Preparation of Crab Green crabs were trapped off the coast of Georgetown, Maine, and transported on ice to the University of Maine (Orono, ME, USA). Live crabs were blast frozen (Southeast Cooler, Lithia Springs, CA, USA) for 1 h at −30 ◦C, then stored at −20 ◦C until use. Frozen whole crabs were thawed for 36–48 h at 4 ◦C before being finely chopped in a Kolsch bowl cutter (UltraSource, Kansas City, MO, USA) and combined with uniodized Kosher salt (Morton Salt, Chicago, IL, USA) at 100 mg g−1, 200 mg g−1, or 300 mg g−1 (w/w). All treatments were prepared separately, in triplicate, and packed into clean, 0.95 L canning jars covered with a double layer of cheesecloth (Pyrm Consumer USA, Spartanburg, SC, USA). 2.2. Fermentation and Sampling Treatments were incubated at 24 ◦C for 120 days with intermittent sampling occurring at 60, 90, and 120 days. On each sampling occasion one entire jar from each treatment replicate was utilized (no repeated sampling). The sauce was separated from the solid residue for testing by straining through two layers of non-sterile cheesecloth into 250 mL centrifuge tubes. The filtrate was centrifuged in an Avanti J-E Beckman Coulter centrifuge (Brea, CA, USA) (100× g, 10 min), and the supernatant was collected for microbial and physicochemical testing. 2.3. Determination of Microbial Activity Crab sauce was serially diluted in 1 g L−1 peptone (Becton, Dickinson, and Co., Sparks, MD, USA) and spread plated in duplicate on skim milk agar (SMA), which consisted of brain heart infusion agar (Hardy Diagnostics, Santa Maria, CA, USA). It was supplemented with 100 mL L−1 (v/v) aseptically packaged skim milk (Natrel, Quebec, Canada) and 30 mg g−1 salt (Aqua Solutions, Deer Park, TX, USA; incubated at 37 ◦C for 48 h) to identify proteolytic bacteria [19]. The brain heart infusion agar was supplemented with 30 mg g−1 salt (incubated at 37 ◦C for 48 h) for total plate count (TPC), and potato dextrose agar (APDA; Alpha Biosciences, Baltimore, MD, USA) was acidified with 0.1 M tartaric acid (Alfa Aesar, Ward Hill, MA, USA; incubated at ambient temperature for 5 day) to isolate Foods 2021, 10, 659 3 of 11 fungi. All plates were counted with colony density between 30 and 300, and the microbial population was expressed as log CFU g−1. 2.4. Determination of Total Volatile Basic Nitrogen (TVBN) and Amine Nitrogen Total volatile basic nitrogen was measured [20] via direct distillation with sodium hydroxide. Lab fermented crab sauce sample was homogenized with trichloroacetic acid (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged (1312× g 20 min). The supernatant was distilled in a micro-Kjeldahl apparatus (Labconco, Kansas City, MO, USA) with sodium hydroxide (Fisher Chemical, Fair Lawn, NJ, USA) and antifoaming agent A (Sigma Aldrich, St. Louis, MO, USA). The distillate was collected in boric acid solution (JT Baker, Center Valley, PA, USA) containing methyl-red (Fisher Scientific, Waltham, MA, USA), a methylene- blue (Sigma Aldrich, St. Louis, MO, USA) indicator, and titrated for a color change with 0.1 N hydrochloric acid (Fisher Scientific, Waltham, MA, USA). TVBN content was expressed as mg 100 mL−1. Proteolytic activity drives increased amine nitrogen content [21]. This measurement is typically used to determine the degree of proteolysis due to activity of endogenous and microbial proteases and can be used to estimate the progress of fermentation. The amine nitrogen was determined using a formol titration published by [22], with the following slight modifications.